**This webpage was produced for an undergraduate course at Davidson College**

A Review of "Synthetic Gene Networks That Count"

Introduction

Synthetic
biology uses many tools from other disciplines, or seeks to imitate
those tools within biological systems. The researchers in this
case wanted to create a synthetic gene network that could be
"programmed" to count to two or three like digital circuits can.
Possible applications of this ability are being able to program
cell death after a certain number of events, or knowing how many times
a certain cellular event occurs. The researchers designed and
built counters using two different methods: using regulation of
the ability of the ribosome to bind to mRNA, and using enzymes that
recombine DNA in a known way.

Riboregulated Transcriptional Cascade (RTC) Counters

Method

The counter constructs were created on plasmids that was then inserted into a strain of E. Coli.
The two-counters and three-counters were built with the same
general method, differing only in how many riboregulated units were
involved in the counter set-up, the two-counter had 2 and the
three-counter had 3 units. The general unit had, in order, a
promoter, a cis-repressor (cr) sequence, a ribosome-binding site (RBS),
and a gene. The cr sequence and the RBS were complentary, so when
the unit was transcribed, a loop would form as the cr sequence and RBS
bound together, blocking the ribosome from landing on the RBS and
starting translation. This loop could be undone by a taRNA
binding to the cr, freeing the RBS for the ribosome to bind to,
starting translation.

Using these facts, the three-counter was designed to work like this:(1) The cell starts with cr-RBS-T7 RNAP transcripts in it(2) First pulse of arabinose binds to BAD promoter which transcribes the taRNA(3) taRNA binds to cr of transcripts, allowing the ribosome to bind to RBS and express T7 RNAP(4)Pulse ends, arabinose and taRNA are degraded, and T7 RNAP expression ends(5) Already translated T7 RNAP transcribes cr-RBS-T2 RNAP(6) Second arabinose pulse creates more taRNA(7) taRNA binds to cr and frees RBS for ribosome binding and T3 RNAP expression(8) Pulse ends, arabinose and taRNA are degraded, and T3 RNAP expression ends(9) T3 RNAP already translated transcribes cr-RBS-GFP(10) Third pulse of arabinose induces more taRNA(11) taRNA binds to cr, freeing RBS for ribosome binding, and GFP is expressed

The
end result of this design should be a cell that does not fluoresce
until after the 3rd arabinose pulse The two-counter uses the same
design only without the middle, T3 RNAP step, thus requiring only 2
pulses to express GFP.

Results

The
experimental results supported the conclusion that the counter design
worked. Cells not given any arabinose did not show any increase
in mean fluorescence level. Those given less then a the full
amount, whether one pulse for the two-counters or either one or two
pulses for the three-counters, showed some increase in fluorescence,
but none matched the increase that occurred in cells that received full
dosages of arabinose. The limited increase in fluorescence for
those cells not getting full doses was explained by the intended
protein being expressed, but the cell also expressing a few proteins
father down the chain than intended, resulting in fluorescence before
it should have occurred.

Model Counters

Methods & Results

Using
the design of their RTC counters, the authors built a mathematical
model of the counters. They then used these models to make
"predictions" for what the experimental results should have been, and
analyzed how closely they matched the actual experimental results.
The predicted results from the model closely matched those of the
real experiments.

Once they authors were confident the model
effectively represented what would actually occur, they used it to test
the effects of varying the pulse duration and time between pulses.
These expected results were also checked against actual results
from experiments that varied the pulse duration and gap between them,
and again the model closely matched the real-world results. This
allowed the researchers to find the maximum and minimum pulse length
and frequency, beyond which the counter could not correctly count due
to kinetic limits of the cellular processes involved.

DNA Invertase Cascade (DIC) Counters

Method

The DIC counters were built using well-known and understood DNA recombinases Cre and flpe
built into units called single invertase memory modules (SIMMs).
Stringing together different numbers of these SIMMs on a plasmid
allow the cells to count to either two or three depending on how many
SIMMs the plasmid includes. The design of the three-counter is 2
SIMMs followed by the GFP gene. The 1st SIMM is the gene for flpe recombinase led by an inverted arabinose-activated promoter, and flanked by sites where flpe can cut, invert the DNA segment, and reattach in the new orientation. The 2nd SIMM is similar except being the cre
gene and being flanked by the restriction sites for Cre, but with the
same inverted arabinose-activated promoter. The last unit is just
the gfp gene whose expression will create fluorescence within the cell. The counter is designed to work like this:(1) The first arabinose pulse expresses flpe(2) flpe cuts, flips, and reattaches the whole first SIMM so that now a correctly orientated promoter is leading the next SIMM(3) This flipping stops flpe transcription and expression because of the gene's inverted orientation with its upstream promoter(4) Second pulse of arabinose transcribes and expressescre (5) Cre cuts, flips, and reattaches the whole second SIMM to place an uninverted promoter upstream of gfp(6) Cre expression ends just as flpe expression ended earlier(7) Third arabinose pulse expresses GFP and the cell begins to fluoresce

The
two-counter design is identical except without the middle Cre-based
step, and thus requiring only two arabinose pulses to express GFP.

The scientists also created another counter by replacing the three arabinose-activated promoters with three different promoters.

Results

The
results for these types of counters mirrored those of the RTC counters,
those cells received some pulses, but not a full dose had a limited
amount of fluorescence, while those receiving a full dosage had a much
higher level of fluorescence. This went for both the two and
three-counters, as well as being true for both those with only the
arabinose-activated promoters and those with the three unique
promoters. The explanation for this limited fluorescence is the
same as that posed for the RTC counters, that a single pulse expressed
the desired protein and a few subsequent proteins.

Figures

Figure 1Figure
1A- This is a pictorial representation of the RTC two-counter design on
the top and the expected results below that. The design shows the
arabinose-activated promoter (P-BAD) upstream of the taRNA, the
P-Ltet0-1 promoter-controlled unit with the cis-repressor (cr),
ribosome binding site (RBS), and the T7 RNA polymerase gene, and the T7
RNAP controlled unit with a cis-repressor, ribosome binding site, and
the GFP gene. The flat headed arrows show the repression
interactions occuring, while the normal arrows show the activation
interactions. The cr represses the RBS, but the taRNA represses
that interaction by binding to the cr, freeing the RBS to be bound to
by the ribosome. The gene product for each preceding step
transcribes the mRNA for the following unit (the pointed arrow).

The
second part of figure 1A pictorial shows what should be the progression
to the end point of the counter. The cell starts with T7RNAP
mRNAs already transcribed (column 0), so the first arabinose pulse
allows the translation of T7 RNAP and its binding to the T7 promoter to
transcribe GFP mRNA (column 1). The second arabinose pulse should
allow the GFP mRNA to be translated, and make the cell fluoresce.

Figure
1B- This shows the actual experimental results for the RTC two-counter.
The two gray-shaded areas represent when the two arabinose pulses
occurred. As can be seen from the graph, those cells getting a
single pulse showed some fluorescence, but those receiving both had
much high fluorescence levels, illustrating that the counters worked.

Figure
1C & D- These serve the same functions as A & B, except for the
three-counter instead. The design pictures (C) show the
additional step inserted in order to count one higher, but the
conventions are the same as A. 1D follows the same conventions as
1B, and the results are simliar in that those cells getting less
than a full dose had some fluorescence, but could not match that of the
cells receiving 3 pulses.

Figure 2

Figure
2A- This is a graph that compares the expected results for the RTC
two-counter from the model to the actual experimental results.
Each line represents the cells that got that pulse, and the shows
the level of fluorescence for that category versus time. The dots
are the experimental values measured at specific time points in the
actual experiment. The color coding is consistant between the
expected and actual lines or dots. The model's predictions
closely mirror those of the actual experiment, showing that the model
is an accurate one.

Figure 2B- This figure is the same type of
figure as 2A, except this one is for the RTC three-counter. Again
we see a close match between the predicted values and the experimental
ones, as well as the relationships between the different categories
being similar.

Figure 2C- The researchers varied the length of
the pulses and the time between pulses within their three-counter
model, and this figure is a graph of the predicted fluorescence levels
as both of those changed. After predicted the results, the
authors preformed the actuall experiment and the solid circles are the
results of that experiment, following the same color scale as used for
the model predictions. These confirmed the model's representation
of the counter's ability to function effectively within a relatively
large range of values.

Figure 2D- This is the same type of graph
as 2C, except what is graphed is the difference in fluorescence between
the three-counter and the two-counter at the same conditions. The
lines are still predictions for the model, and the solid circles are
actual experimental results. This figure further confirmed the
ideal range for effective counting using this design.

Figure 3

Figure
3A- This is a pictoral representation of the second type of counter,
the DIC counter. The individual units that make up the whole
counter are the SIMMs, and one is outlined by the dashed box. A
SIMM includes an inverted arabinose-activated promoter (P BAD), a
RBS, a recombinase gene, a tag for quick degradation of the protein
(ssrA), and a transcriptional terminator (Term), all flanked on either
side by the cut sites specific for the recombinase encoded within the
unit. This specific diagram is for the three-counter and has two
SIMMs plus the GFP gene at the end, and uses the flpe, with its FRT cut
sites, and the Cre, with its lox cut sites, as the recombinases.
The first arabinose pulse transcribes the first SIMM, which is
then expressed, cutting and flipping the whole unit at the FRT sites.
The second pulse does the same thing for the second SIMM, which
cuts at the lox sites instead, but still flips the unit. The
third and final pulse transcribes and translates GFP. Each flip
correctly orients the P BAD for the transcription of the next unit in
the process.

Figure 3B- This is a plot of the experimental
results for the DIC three-counter. You can see the small
increases in fluorescence in the first 2 pulses due to premature
activation of the later process steps, but the increase that comes with
the third pulse is by far the largest increase, as it should be if the
design functions properly. The shaded regions show the arabinose
pulses so that the reader can follow where the counter should be in its
process.

Figure 3C- Again the researchers created a mathematical
model of their counter design, and used that model to predict the
effects of varying pulse length and the time between pulses. What
they graphed however, is the ratio of fluorescence levels between the
three-counter and the two-counter. The black dots on the graph
are experimentally determined results, which closely match the model
values. The graph shows ideal ranges of pulse lengths and
intervals for the counter to effectively count, which is an important
thing to consider when considering uses for the design.

Figure 4

Figure
4A- This is a picture of the DIC counter design that is the same as
that shown in Figure 3A, except that this one uses 3 different
promoters instead of using P BAD for all of them. In this set-up,
the first pulse is anhydrotetracycline (aTc), the second pulse is
arabinose, and the third pulse is isopropyl
beta-D-1-thiogalactopyranoside (IPTG), each of which activates the
promoter for that step, aTc for P Ltet0-1, arabinose for P BAD, and
IPTG for P A1lac0.

Figure 4B- This is a graph of flow cytometry
data, which measures the level of fluorescence in cells. Every
possible combination of the order of the three pulses was tested and
the results are shown in this figure. Only those cells that
received pulses of aTC, arabinose, and IPTG in that order, had
substantial levels of fluorescence at the end of the third pulse,
supported the conclusion that the counter design was properly
functional.

Figure 4C- This figure is the flow cytometry data
comparing those cells that received a single pulse of any of the three
activators to those that received all three in the correct order.
As can be seen in the graph, only the cells receiving all three
pulses had a large number of cells with a high level of fluorescence.
The others are all grouped together where cells with low
fluorescence fall.

Figure 4D- This figure is similar to 4C, but
this time all the cells that received any two pulses are compared to
those that received all three in the correct order. Again those
cells that got the full dosage have much higher levels of fluorescence
than those that only got two of the pulses. This further
supports the counter's design functioning as intended.

Opinion

I
found this paper and what they were able to make the cells do very
interesting, as did friends that I explained it to when asked what I
was working on. I wish there had been more explanation for why
they performed the N-(N-1) (Figure 2D) analysis, or at least why they
included it in the paper over other figures, as I had some trouble
understanding what that graph contributed that was not shown in Figure
2C. Especially as the experimental values in Figure 2D only
seemed to match the model's predictions in a few places, not as well as
they matched for other figures. They also showed the level of
fluorescence in multiple different ways between or even within figures.
In Figures 1 & 2 the fluorescence is in arbitrary units, but
for Figures 3 & 4 it has been normalized to the highest one.
The within Figure 4, C & D just show the fluorescence at the
end of the process, whereas they've shown how it changed with time for
all the cells getting 3 pulses in B. I understand splitting the
one, two, and three pulse categories onto different figures so they can
be seen better, but why not use the style of B for all three since you
can see the final fluorescence level as well as how it changed with
time. The paper certainly supports the fact that they created
cells that could count to two or three, I just was thrown by the
mismatching of some of the figures.